Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes.

Abstract

BACKGROUND:

Hypoxic culturing of chondrocytes is gaining increasing interest in cartilage research. Culturing of chondrocytes under low oxygen tension has shown several advantages, among them increased synthesis of extracellular matrix and increased redifferentiation of dedifferentiated chondrocytes. Quantitative gene expression analyses such as quantitative real-time PCR (qRT-PCR) are powerful tools in the investigation of underlying mechanisms of cell behavior and are used routinely for differentiation and phenotype assays. However, the genes used for normalization in normoxic cell-cultures might not be suitable in the hypoxic environment. The objective of this study was to determine hypoxia-stable housekeeping genes (HKG) for quantitative real-time PCR (qRT-PCR) in human chondrocytes cultured in 21%, 5% and 1% oxygen by geNorm and NormFinder analyses.

RESULTS:

The chondrocytic response to the hypoxic challange was validated by a significant increase in expression of the hypoxia-inducible gene ankyrin repeat 37 as well as SOX9 in hypoxia. When cultured on the 3-dimentional (3D) scaffold TATA-binding protein (TBP) exhibited the highest expression stability with NormFinder while Ribosomal protein L13a (RPL13A) and beta2-microglobulin (B2M) were the most stable using geNorm analysis. In monolayer RPL13A were the most stable gene using NormFinder, while geNorm assessed RPL13A and human RNA polymerase II (RPII) as most stable. When examining the combination of (3D) culturing and monolayer RPL13A and B2M showed the highest expression stability from geNorm analysis while RPL13A also showed the highest expression stability using NormFinder. Often used HKG such as beta actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the most unstable genes investigated in all comparisons. The pairwise variations for the two most stable HKG in each group were all below the cut-off value of 0.15, suggesting that the two most stable HKG from geNorm analysis would be sufficient for qRT-PCR.

CONCLUSION:

All data combined we recommend RPL13A, B2M and RPII as the best choice for qRT-PCR analyses when comparing normoxic and hypoxic cultured human chondrocytes although other genes might also be suitable. However, the matching of HKG to target genes by means of a thorough investigation of the stability in each study would always be preferable.

Expression of ANKRD37 (A) SOX9 (B). Relative mean expression is presented on day 1, 2, and 6 in human chondrocytes cultured in 21%, 5% or 1% oxygen (error bars: SEM). RPL13A and B2M are used as reference genes. Values for each sample are normalized to baseline expression.

Average expression stability measure (M) of candidate HKG. During stepwise exclusion of the least stable gene in monolayer, 3D culturing in MPEG-PLGA scaffolds, and the combined stability for all samples M is calculated.

Pairwise variation (V). V between two sequential normalization factors containing an increasing number of genes in monolayer, 3D culturing in MPEG-PLGA scaffolds, and the combined stability for all samples is calculated.